The characteristics of the proximal tubular Na+-H+ antiporter were determined in isolated proximal tubular cells to ascertain whether the features of this transport system in intact cells are comparable with those previously described for isolated brush-border membrane vesicles. A method is described for the rapid isolation of a purified preparation of cells that demonstrate morphological and functional characteristics of the renal proximal tubule. The cells maintain their polarity while in suspension, and adenylate cyclase activity is enhanced by parathyroid hormone but not by arginine vasopressin. The cells display gluconeogenic function and Na+-dependent alpha-methyl-D-glucose and organic phosphate cotransport, processes that confirm their proximal tubule origin. O2 consumption rates and cytosolic adenosine triphosphate levels indicate functional integrity. Na+-H+ antiport activity was defined in these cells by measuring amiloride-sensitive Na+ uptake. At intracellular pH = 6.4 vs. extracellular pH = 7.4, KtNa was 10.1 +/- 2.8 mM, and maximal sodium flux was 0.89 +/- 0.13 nmol X 10(6) cells-1 X K0.5 for amiloride and ethyl-isopropyl amiloride, measured at an external Na+ concentration of 1 mM, was observed at 2.5 X 10(-5) M and 2.9 X 10(-6) M, respectively. The external and internal loci of the exchanger displayed asymmetric affinity for the hydrogen ion: the apparent pK for the external site was 7.20-7.26 vs. less than 6.5 for the internal site. The internal site demonstrated features of positive cooperativity. In summary, the Na+-H+ antiporter present in the luminal membrane of the renal proximal tubule has been characterized in the intact cell and displays functional and kinetic parameters closely resembling those described in isolated brush-border membrane vesicles.
The affinity for Na+ of the cytoplasmic vs. external transport site of the amiloride-sensitive Na+-H+ antiporter was studied in confluent cultures of MDCK cells. Na+-H+ antiport activity was fluorometrically determined by monitoring changes in intracellular pH (pHi) using the pH-sensitive fluorescent probe, BCECF. Na+-dependent H+ fluxes were studied both in the functionally operative (H+ efflux/Na+ influx) and reverse (H+ influx/Na+ efflux) mode of antiport activity, under pH equilibrium, but Na+-gradient conditions. Thus the driving force for antiport activity was solely dependent on the transmembrane Na+ gradient. Independent experiments established that pHi and intracellular Na+ [Na+i] had been set at the desired values before the initiation of a particular experiment. Under conditions of pHi = pHo = 7.0, [Na+i] = 0 mM and varying extracellular Na+ concentration [Na+o], the apparent affinity for Na+ (KtNa) for the external transport site was 24 +/- 3 mM. When antiport activity was measured in the reverse mode of operation, but under identical pH conditions, KtNa at the internal site was 7 +/- 1 mM. When ambient pH was elevated to 7.5, KtNa at the internal site was 14 +/- 1 mM. Maximum H+ flux (JmaxH+) for the antiporter under all three conditions was not significantly different. In summary, the Na+-H+ antiporter displays asymmetric affinity for Na+ at the internal vs. external transport site. Under pH equilibrium conditions, the affinity of the Na+-H+ antiporter for Na+ is three- to four-fold greater at the internal vs. external locus, and the affinity for Na+ at the internal site is enhanced by lower pHi. The close similarity between values for KtNa (inside) and reported values for intracellular Na+ concentration suggests that regulation of the Na+-H+ antiporter may be affected by changes in intracellular Na+ concentration.
The renin-angiotensin-aldosterone system was studied in fifty healthy children aged 4-16 years under normal sodium and potassium intake. The plasma renin activity (PRA) and plasma aldosterone (PA) decreased with age: r = -0.30, P less than 0.05 for plasma renin activity and r = -0.33, P less than 0.05 for plasma aldosterone. Significant negative correlation was obtained between plasma renin activity and the 24-h urinary sodium excretion; r = -0.40, P less than 0.01. This relationship remained significant when the daily urinary sodium excretion was corrected for 1.73 m2 body surface area (BSA); r = -0.40, P less than 0.01. Using the multivariance analysis, plotting the plasma renin activity against the two combined parameters (24-h urinary sodium excretion and age), no improvement was obtained (r = 0.38, P greater than 0.05). This finding suggests that during childhood, sodium rather than age has a major modulatory role on plasma renin activity. With advancing age the plasma aldosterone showed a significant positive correlation coefficient with plasma renin activity(r = 0.29, P less than 0.05). Multivariance analysis between plasma aldosterone and the two combined parameters, Plasma renin activity and age, significantly improved the correlation coefficient (r = 0.42, P less than 0.05) suggesting that both plasma renin activity and age play a dominant modulatory role in the control of plasma aldosterone during childhood. Neither 24-h urinary sodium excretion, nor 24-h urinary potassium excretion, improved the multiple correlation coefficient with plasma aldosterone when added to plasma renin activity and age.
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